This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Some O-linked protein modifications are reversible post-translational modifications of paramount importance in biological signaling pathways and are found in substoichiometric concentrations in the cell. This fact and the lability of these modifications under collisional activation conditions in the mass spectrometer make very difficult their detection by conventional tandem MS strategies. Several methods for detecting these modifications have been devised, including derivatization and labeling strategies. However, all these methods suffer from insufficient sensitivity. Derivatization methods using beta-elimination followed by Michael additions address this problem but intorduce two new problems: i) phosphorylation and glycosylation compete for the same site and the conditions for beta-elimination are difficult to fine-tune such that only one modification is derivatized and not both;ii) derivatization methods remove the glycan and thus do not differentiate between complex O-glycosylation or single O-GlcNAc additions. In order to detect this extremely labile modification by MS, both the ionization methods and fragmentation methods used during analysis have to be extremely "soft" to keep the modification intact. Two instruments developed in this laboratory (vibrationally cooled MALDI FTMS and ESI-qQq FTMS) have proven to be ideal for both O-glycosylation and phosphorylation analysis as they both provide very soft ionization conditions and the ion optics are tunable such that the modified proteins/peptides are transferred the ICR cell intact. Electron Capture Dissociation (ECD) is the method of choice for inducing peptide fragmentation as it does not cleave the modifications from the peptide side chains. We have verified that ECD has the remarkable ability to extensively fragment peptides yielding almost complete sequence information (with the exception of proline) while not causing fragmentation within labile side-chain modifications including phosphorylation and O-GlcNAc. In addition, we have investigated the use of low-energy SORI-CAD for fragmenting and sequencing O-GlcNAc and O-phosphorylated peptides. Under carefully selected conditions, SORI-CAD can also result in peptide backbone fragmentation leaving the modification intact. The O-GlcNAc modification is extremely interesting as it occurs in the nucleus and cytoplasm, appears to be as common as phosphorylation, and is involved in protein signaling. Proteins that carry this modification, such as the C-terminal domain of RNA polymerase II exhibit either the phosphate or the O-GlcNAc modification, and these modifications govern the state of activity of the protein (Yin-Yang theory). This study is therefore biologically relevant and the methods we have devised to carry out proteomic studies are likely to have a high impact on the field. We have presented some results at meetings and are preparing a manuscript on the analysis of O-GlcNAc-modified peptides. We have also undertaken studies of O-linked glycans using CID and C-trap dissociation on the LTQ-Orbitrap tandem MS and Electron Transfer Dissociation (ETD) on the Bruker quadrupole ion traps.